The Toll-like receptors (TLRs) comprise a family of at least 11 integral membrane receptors that are essential for initiating inflammatory reactions in response to pathogens such as viruses and bacteria. The major objective of this research is to elucidate the structures of external domains of TLRs in order to learn how they interact with pathogen associated molecular patterns (PAMP). In this past year we have developed a baculovirus expression system for the TLRs in conjunction with the Protein Expression Lab [SAIC] and Joseph Shiloach, head of the NIDDK Biotechnology Unit. The purification scheme for the target TLRs utilizes a double affinity tag and typically yields 10-15 mg of highly purified sample/10L prep. Biochemically, the stability and glycosylation states have been analyzed for the first two TLRs purified. Ligand binding studies for one TLR have been initiated both in the Davies laboratory by gel filtration and ITC experiments and in collaboration with the Margulies laboratory in NCI using surface plasmon resonance. Finally, crystals and initial diffraction data have been collected for one of the TLRs. Currently, we are searching for suitable heavy atom derivatives to solve the phase problem